The generation of long-lived, high affinity antibodies (Ab) is required for protective immunity to most viruses and for protection after vaccination. Thus, it is essential to understand the mechanisms that control the generation of long-lasting protective Ab responses. T follicular helper (Tfh) cells, a distinct CD4+ T cell subset that expresses high levels of CXCR5 and localizes in the B cell follicles, play an essential role on promoting long-lived Ab responses. In fact, in the absence of Tfh cells, long-term Ab responses are impaired and protection to pathogens compromised. Therefore, it is essential that we understand how to manipulate Tfh responses in order to improve the efficacy of vaccines. However, despite significant advances in the field, our understanding of how Tfh cells responses are initiated is very limited. Recent studies suggest that Tfh cells are initially primed by dendritic cells (DCs), suggesting that we may be able to develop adjuvants that preferentially activate DCs to promote Tfh cell priming, or target vaccine antigens to those DCs that preferentially induce Tfh cells. Unfortunately, we do not know what signals direct the DCs to promote Tfh cell differentiation or which specific subsets of DCs prime Tfh cell responses. Thus, the long-term goal of this proposal is to determine how virus-specific Tfh cells are primed by DCs, which will help us to determine the nature of adjuvants that can be used to boost Tfh cell responses. Importantly, whereas some studies suggest that Tregs, particularly the T follicular regulatory (TFR) cells, suppress Tfh and GC B cell responses, we recently found that Tregs promote Tfh and B cell responses to influenza. Mechanistically, Tregs favored influenza-specific Tfh cell development by limiting the physiological availability of IL-2, a potent suppressor of Tfh cell differentiation. Thus, in addition to DCs, Tregs are also required for normal Tfh cell responses. The central hypothesis that will be tested in this proposal is that Tfh cells are primed within particular low-IL-2 microenvironments outside the T cell area, where coordinated encounters between pro-Tfh DCs (which provide pro-Tfh signals) and Tregs (which consume IL-2) allow Tfh cell differentiation. To test this hypothesis, in Aim 1 we will first identify the specific DC subsets and activation state of DC that is required to prime Tfh cell responses, and the molecular mechanisms by which DC/Tregs/IL-2 interplay regulates this process. In Aim 2 we will determine the chemokine-chemokine receptor interactions that orchestrate pro-Tfh DC and Tregs encounters outside the T cell zone. In Aim 3 we will determine how IL-2 and other factors control the development of TFRs (which inhibit Tfh cell responses), so we can prevent TFR differentiation after vaccination. We believe that our work will significantly contribute to our understanding of how Tfh cell responses are initiated and reveal new strategies to elicit long-lived Ab responses to infection and vaccination. This proposal is innovative because it focuses on previously ignored, yet profoundly important, aspects of Tfh cell biology.